Understanding the effects of alcohol on brain function is critical for developing better treatments for alcohol- related problems. In this application to extend an ongoing research project, we focus on understanding how alcohol affects the function of a key ion channel expressed by neurons. This channel, the N-methyl-D- aspartate (NMDA) receptor, plays a major role in excitatory glutamatergic transmission and is critically involved in complex processes that underlie learning, memory and other higher cognitive processes. Previous studies carried out during this project established that alcohol's inhibition of the NMDA receptor could be modulated by discrete domains on the receptor and we identified key residues within transmembrane domains of the protein that may define an alcohol site of action. We used this knowledge to generate a novel knock-in mouse that shows reduced sensitivity to the acute actions of ethanol. In this application, we propose a series of specific aims designed to further understand how transmembrane domains control the receptor's sensitivity to alcohol and how expression of ethanol-insensitive receptors in vivo affects alcohol-induced behaviors and drinking. Aim 1 will test the hypothesis that specific amino acids within sub-domains of the NMDA receptor determine the ability of ethanol to inhibit receptor function. Experiments in this aim will use recombinant expression and recording techniques to test how site-directed mutagenesis of residues in key transmembrane domains including TMl and pre-TM4 alters the effects of alcohol on channel function. Aim 2 continues studies investigating how phosphorylation affects the ethanol sensitivity of NMDARs and examines how different co-agonists alter ethanol inhibition. Aim 3 studies will test the hypothesis that the behavioral responses to alcohol can be modified by expression of mutant NMDA receptors that show altered ethanol sensitivity. Experiments in this aim will use a newly created GluN2A knock-in gene construct to generate knock-in mice with reduced sensitivity to ethanol. Overall, results from these studies are expected to lead to a more complete understanding of the role of NMDA receptors in mediating alcohol's action of the brain.